1. Field of the Invention
This invention relates to novel monoesters of phosphoric acid and more particularly, novel phosphoric esters which are hydrolyzed by the action of an alkaline phosphatase to release an azo dye and a method for the analysis of the alkaline phosphatase, using the ester as a substrate.
It is very important for a clinical examination to see the activity of an alkaline phosphatase in human body fluid. It is well known that when the activity of an alkaline phosphatase is high, metabolism in the liver, bones, or the like is generally under the unfavourable conditions and therefore, symptons of liver disease, rickets, osteosarcoma, dysthyroidism, or the like appear. Accordingly, it is quite, natural that various methods for the determination of an alkaline phosphatase activity have been proposed.
It is required that a method for the quantitative determination of an alkaline phosphatase used in an ordinary clinical examination is simple in operation and has a good reproducibility. For this purpose, there are so-called a colorimetry and a fluorometry.
The colorimetry is a method of quantitative analysis in which a compound which is hydrolyzed by the action of an alkaline phosphatase to release a dye or a dye-precursor is used as a substrate of an enzymatic reaction, and the dye released in the enzymatic reaction is colorimetrically measured or the dye-precursor released in the enzymatic reaction is reacted with a reagent thereby to be converted into the dye which is then colorimetrically measured to determine the activitiy of the alkaline phosphatase. The colorimetry has an advance that a colorimeter or a spectrophotometer which are widely spread may be conveniently utilized to conduct the analysis. On the other hand, the fluorometry is a method of quantitative analysis in which a compound which is hydrolyzed by the action of an alkaline phosphatase to release a fluorescent substance is used as a substrate of an enzymatic reaction, and a fluorescent intensity of the fluorescent substance released in the enzymatic reaction is measured to thereby determine the activity of the alkaline phosphatase. The fluorometry has a higher sensitivity as compared with the colorimetry, however it has some disadvantages that it is apt to be disturbed by a trace of fluorescent substance which may coexist therewith and a fluorometer has not so widely spread.
This invention relates to novel mono-4-arylazoaryl phosphoric esters which may be used as a suitable substrate in a method for the quantitative analysis of an alkaline phosphatase by a colorimetry.
2. Description of the Prior Art
In the past, there have been used the following methods for the quantitative analysis of an alkaline phosphatase by a colorimetry.
(1) The method in which disodium p-nitrophenyl phosphate is used as a substrate and p-nitrophenol which is released on hydrolysis of the substrate by the action of an alkaline phosphatase is colorimetrically measured to determine the activity of the alkaline phosphatase, which is specifically described in O. A. Bessey, O. H. Lowry and M. J. Brock, "Journal of Biological Chemistry", Vol. 164, p. 321 (1946).
(2) The method in which phenolphthalein phosphate is used as a substrate and phenolphthalein which is released on hydrolysis of the substrate by the action of an alkaline phosphatase is colorimetrically measured to determine the activity of the alkaline phosphatase, which is specifically described in A. L. Babson, S. J. Greeley, C. M. Coleman, and G. E. Phillips, "Clinical Chemistry", Vol. 12, p. 482 (1966).
(3) The method in which thymolphthalein phosphate is used as a substrate and thymolphthalein which is released on hydrolysis of the substrate by the action of an alkaline phosphatase is colorimetrically measured to determine the activity of the alkaline phosphatase, which is specifically described in C. M. Colemann, "Clinical Chemistry", Vol. 13, p. 401 (1966).
(4) The method in which thymolblue mono-phosphate is used as a substrate and thymolblue released on hydrolysis of the substrate by the action of an alkaline phosphatase is colorimetrically measured to determine the activity of the alkaline phosphatase, which is specifically described in Japanese Patent Disclosure (OPI) No. 136662/1976.
(5) The method in which phenyl phosphate is used as a substrate and phenol released on hydrolysis of the substrate by the action of an alkaline phosphatase is oxidatively condensed with 4-amino-antipyrine in the presence of potassium ferricyanide to form red quinone which is colorimetrically measured to determine the activity of the alkaline phosphatase, which is specifically described in P. R. N. Kind and E. J. King, "Clinical Pathology", Vol. 7, p. 322 (1954); K. Watanabe et al., "Rinsho-Byori or Clinical Pathology", Vol. 15, p. 708 (1967); T. Nakayama et al., "Rinsho-Byori or Clinical Pathology", Vol 23 (a supplementary volume in the general meeting), p. 85 (1975).
Among the methods in the prior art described above, the method (1) is most widely used as a method for the quantitative analysis of an alkaline phosphatase, however, the colorimetry is carried out at 410 nm and therefore, this method is interfered with a colored substance such as bilirubin, hemoglobin, etc. contained in serum and thus, it requires a blank test which makes operations more complicate. The prior methods (2) to (5) tentatively overcome the disadvantage of the method (1) because a colorimetry in the methods (2) to (5) is conducted at a longer wavelength region than that in method (1). However, it is difficult to synthesyze highly pure mono-phosphoric esters of phthaleins which are used as a substrate in the methods (2) to (4). In other words, these mono-phosphoric esters may be obtained by hydrolysis of monophosphorodichloridate of corresponding phthalein which may be prepared by reacting one hydroxyl group of the phthalein molecule with phosphorous oxychloride. In this reaction, a higher molar ratio of phosphorous oxychloride to phthalein causes phosphorylation of more than one hydroxyl group, while a lower molar ratio thereof causes formation of two or three kinds of products since one molecule of phosphorous oxychloride reacts with two or three molecules of phthalein. In either case, an unsuitable compound for a substrate to be used in the quantitative analysis of an alkaline phosphatase is produced as a secondary product. Accordingly, the isolation and purification of the product after the reaction require a troublesome operation and as the result, it is difficult to obtain products having a high degree of purity. On the other hand, the method (5) of the prior art comprises the two step reactions of an enzymatic, phenol-release reaction and subsequent coloring reaction, which require complicated operations.
Thus, it has been expected that an improved method for the quantitative analysis of an alkaline phosphatase without the disadvantages described above would appear.